1. During the characterization of recombinant CYP2C19, it was observed that this enzyme metabolized midazolam, which is generally regarded as CYP3A4/5 substrate, and we therefore decided to pursue this observation further. 2. CYP2C19 showed a Michaelis-Menten pattern for midazolam 1'-hydroxylation and was inhibited by (+)-N-3-benzylnirvanol and S-mephenytoin, which are a standard potent inhibitor and a substrate of CYP2C19, respectively. 3. The inhibitory potency by CYP3A4/5 inhibitor on the midazolam 1'-hydroxylation in human liver microsomes (HLM) was correlated with the CYP3A4/5 specific catalytic activity, but such correlation was not observed in CYP2C19 enzyme. The in vitro intrinsic clearance value for midazolam 1'-hydroxylation was not changed by the addition of (+)-N-3-benzylnirvanol in four individual HLM preparations. 4. These results indicated that although CYP2C19 is capable of catalyzing midazolam 1'-hydroxylation, CYP3A4/5 play a more important role.